The overall goal of this proposed research is to develop a second generation Imaging MALDI mass spectrometer with significant performance advancements, to develop new procedural and chemical methodologies for automated sample preparation, and apply this new instrumentation and methods to several biological research investigations that will significantly benefit from this technology. Instrumental advancements will be done to a current Applied Biosystems Voyager STR MALDI MS for high speed imaging, including installation of a 1 kHz laser, modified electronics, new multi-anode detector, a 2-5 micron laser spot source, an X, Y, Z sample movement stage of 0.2 micron precision, and software for data acquisition and data processing including building a tissue protein database with appropriate archival and search routines. The second aspect of the proposed work is the establishment of new sample preparation methods to improve application of matrix in terms of the ease of use and reproducibility of the process, employment of different surface chemistries for tissue blotting, synthesis of cleavable detergents that enhance the MALDI process, evaluation of tissue fixing methods, construction and testing of a robotic picoliter droplet dispensing robot, new methods to improve relative quantitation of proteins on tissues, and methods designed for the imaging of small molecules such as drugs and drug metabolites by MALDI MS/MS. Concurrent with the instrument development would be the application of the technology to several biological problems that would significantly benefit from profiling and imaging mass spectrometry. These include a detailed molecular mapping of mouse epididymis, imaging drugs and drug metabolites involved in CNS dysfunction and correlation of this with changes in protein profiles, studies of protein profiles comparing normal brain regions with those involving Parkinson's disease, and investigation of changes in protein profiles in human epilepsy. Finally, we propose to develop the technology for the creation of 3- dimensional molecular images of selected proteins of small structures in tissues or organs such as a fetal mouse heart. The latter would focus on developmental aspects of heart formation and growth.